Figure 1
From: The 3D architecture of a bacterial swarm has implications for antibiotic tolerance
Tracking 3D trajectories of fluorescent bacteria in a multilayered swarm using defocused imaging (DI) methodology. (A) A macroscopic view of a S. marcescens swarming colony inoculated in the center of the agar plate and allowed to swarm out. The region observed for tracking was ~100–400 μm behind the advancing edge (downward pointing arrow). (B) A sketch of the cross section of the colony near the edge, illustrating the approximate shape and dimensions in this region. The height of the colony at the point of observation was estimated to be ~40 μm (see Methods). This height was arbitrarily divided into 15 levels or bins. (C) A snapshot of off-focus fluorescent images viewed from the top. The diameters of the diffraction rings report on bacterial location at different heights in the colony. Arrow points to an example of a relatively large diffraction ring corresponding to location of a cell 35 μm above the agar. There are, on average, 1000 times more unlabeled non-fluorescent cells in any view of the fluorescence image as determined by viewing the same field in phase contrast (see Movie S4). (D) The calibration curve correlating the diameter of the diffraction ring as a function of the depth of the cells, as described in Methods. (E) A cartoon showing how height is estimated from the size of the diffraction ring for bacteria in C. (F) The x-y trajectory of a typical cell moving inside the 3D colony. (G) The height of the same cell plotted in F as a function of time. (H) The 3D trajectory of the same cell plotted in (F,G). Small red arrows indicate instantaneous vectors of the torsion and curvature of the trajectory (see Methods).
